Abstract Investigation of erythrocytes from spontaneous or engineered germ‐line mutant mice has been instrumental in characterizing the physiological functions of components of the red cell cytoskeleton and membrane. However, the red blood cell expresses some proteins whose germline loss‐of‐function is embryonic‐lethal, perinatal‐lethal, or confers reduced post‐weaning viability. Promoter regions of erythroid‐specific genes have been used to engineer erythroid‐specific expression of Cre recombinase. Through breeding with mice carrying appropriately spaced insertions of loxP sequences, generation of erythroid‐specific knockouts has been carried out for signaling enzymes, transcription factors, peptide hormones, and single transmembrane span signaling receptors. We report here the use of Cre recombinase expression driven by the erythropoietin receptor (EpoR) promoter to generate EpoR‐Cre;Kcc3f/f mice, designed to express erythroid‐specific knockout of the KCC3 K‐Cl cotransporter encoded by Kcc3/Slc12A6. We confirm KCC3 as the predominant K‐Cl cotransporter of adult mouse red cells in mice with better viability than previously exhibited by Kcc3−/− germline knockouts. We demonstrate roughly proportionate preservation of K‐Cl stimulation by hypotonicity, staurosporine, and urea in the context of reduced, but not abrogated, K‐Cl function in EpoR‐Cre;Kcc3f/f mice. We also report functional evidence suggesting incomplete recombinase‐mediated excision of the Kcc3 gene in adult erythroid tissues.
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